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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
>chr7:45354619-45354713 95bp CCCTCCTCCTATTCCAGGTG AAGGGATGAGAGTTGAGAAGGA
Mutant= 98 bp
Wild Type = 95 bp
Sequence
Wt Sequence: gggcacagtgaggatatctcaggggaggggctaaaatgggggcggggcttagatgaggagccgggttgagtgaGACATGAAGGGTGGGGCTCAACTGAGGATCAGAGTAGTGGGTGGGACTGGTTTGGTTGAGATGAGGGATGGGTTAAGGAGGGACTAAACAGGAATCAAGATGAAGCAAGGCTTAGATGAGGGGCATGATTTCGAACAAGGGGAATGATCTCAGAAAGACCCGCTGGACAGTCTCTGCCCTGTTTCCCTTTGTTCAGGCCCGGCAGAGGGAGAAAATGAACGATGACCACAATAAGCGACTGTCAGAGACGGTGGACAAGCTGCTAAGTGAATCCAACGAACGGTTACAGCTTCACCTCAAAGAACGTATGGGGGCACTAGAGGAGAAGGTGCGCCCCCTCCCACCCCTGCCCCAGACCTTTGTCAACCAGGGGTGGCTCCAAATGCCGTCCTAGGGCCCATACATCTTCAGATCTGTCATTTGAGACCTCCATACCCACAAACTCTGCTCTGTGACTGAAGGCACCATTTTGCTTGGCCATTCCAAGAGCGGAGTTTTCCTAGACCACAACCCCTGCAGCCTGCCTTTGACCTTCCATTTCACCCCTGTCCCCACAGAACTCACTGAGTGAGGAGATTGCTAACATGAAGAAACTTCAAGATGAGCTGCTGCTGAATAAGGTGAGGACCCGGAACTTAATGGGGAGTTATCGAGTGGCCTGTTCCTCATAGCAGTGACATCGAGCCCCATACCTCTGCCTCTCTCCACAGGAGCAGCTCTTGGCTGAGATGGAACGGATGCAGATGGAGATCGACCAGCTCCGGGGAAGGCCGCCCTCCTCCTATTCCAGGTGACAGAAGTGCTTCCTGCCCTTTCTGTCTGTGgcaagctcctcctttccgctcctccttctcaactctcatcccttattagtttatttattttatg
Deleted Sequence: GACATGAAGGGTGGGGCTCAACTGAGGATCAGAGTAGTGGGTGGGACTGGTTTGGTTGAGATGAGGGATGGGTTAAGGAGGGACTAAACAGGAATCAAGATGAAGCAAGGCTTAGATGAGGGGCATGATTTCGAACAAGGGGAATGATCTCAGAAAGACCCGCTGGACAGTCTCTGCCCTGTTTCCCTTTGTTCAGGCCCGGCAGAGGGAGAAAATGAACGATGACCACAATAAGCGACTGTCAGAGACGGTGGACAAGCTGCTAAGTGAATCCAACGAACGGTTACAGCTTCACCTCAAAGAACGTATGGGGGCACTAGAGGAGAAGGTGCGCCCCCTCCCACCCCTGCCCCAGACCTTTGTCAACCAGGGGTGGCTCCAAATGCCGTCCTAGGGCCCATACATCTTCAGATCTGTCATTTGAGACCTCCATACCCACAAACTCTGCTCTGTGACTGAAGGCACCATTTTGCTTGGCCATTCCAAGAGCGGAGTTTTCCTAGACCACAACCCCTGCAGCCTGCCTTTGACCTTCCATTTCACCCCTGTCCCCACAGAACTCACTGAGTGAGGAGATTGCTAACATGAAGAAACTTCAAGATGAGCTGCTGCTGAATAAGGTGAGGACCCGGAACTTAATGGGGAGTTATCGAGTGGCCTGTTCCTCATAGCAGTGACATCGAGCCCCATACCTCTGCCTCTCTCCACAGGAGCAGCTCTTGGCTGAGATGGAACGGATGCAGATGGAGATCGACCAGCTCCGGGGAAGGCCGCCCTCCTCCTATTCCAGGTGACAGAAGTGCTTCCTGCCCTTTCTGTCTGTG
824 bp deletion beginning at Chromosome 7 negative strand position 45,355,486 bp and ending after 45,354,663 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37874 | CCC TCC TCC TAT TCC AGG TG | Wild type Forward | A | |||
| 37875 | AAG GGA TGA GAG TTG AGA AGG A | Common | A | |||
| 37876 | CAG GGG AGG GGC TAA AAT G | Mutant Forward | A | |||
| 37877 | Fluorophore-1 | TGC TTC CTG CCC TTT CTG | Quencher-1 | WT Probe | ||
| 37878 | Fluorophore-2 | CTT AGA TGA GGA GCC GGG TT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37874 | 0.40 uM |
| 37875 | 0.40 uM |
| 37876 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.